The invention relates to modified proteins, and DNAs encoding the same, which are biosynthetic, chemosynthetic or recombinant constructs derived from the TGF-xcex2 superfamily of structurally related proteins, including modified morphogenic proteins.
The TGF-xcex2 superfamily includes five distinct forms of TGF-xcex2 (Sporn and Roberts (1990) in Peptide Growth Factors and Their Receptors, Sporn and Roberts, eds., Springer-Verlag: Berlin pp. 419-472), as well as the differentiation factors Vg-1 (Weeks and Melton (1987) Cell 51: 861-867), DPP-C polypeptide (Padgett et al. (1987) Nature 325: 81-84), the hormones activin and inhibin (Mason et al. (1985) Nature 318: 659-663; Mason et al. (1987) Growth Factors 1: 77-88), the Mullerian-inhibiting substance, MIS (Cate et al. (1986) Cell 45:685-698), osteogenic and morphogenic proteins OP-1 (PCT/US90/05903), OP-2 (PCT/US91/07654), OP-3 (PCT/WO94/10202), the BMPs, (see U.S. Pat. Nos. 4,877,864; 5,141,905; 5,013,649; 5,116,738; 5,108,922; 5,106,748; and 5,155,058), the developmentally regulated protein Vgr-1 (Lyons et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4554-4558) and the growth/differentiation factors GDF-1, GDF-3, GDF-9 and dorsalin-1 (McPherron et al. (1993) J. Biol. Chem. 268: 3444-3449; Basler et al. (1993) Cell 73: 687-702), to name but a few.
The proteins of the TGF-xcex2 superfamily are disulfide-linked homo- or heterodimers that are expressed as large precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved N-terminal pro region sequence of several hundred amino acids, a cleavage site, a mature domain comprising an N-terminal region that varies among the family members and a highly conserved C-terminal region. This C-terminal region, present in the processed mature proteins of all known family members, contains approximately 100 amino acids with a characteristic cysteine motif having a conserved six or seven cysteine skeleton. Although the position of the cleavage site between the mature and pro regions varies among the family members, the cysteine pattern of the C-terminus of all of the proteins is in the identical format, ending in the sequence Cys-X-Cys-X (Sporn and Roberts (1990), supra).
A unifying feature of the biology of the proteins of the TGF-xcex2 superfamily is their ability to regulate developmental processes, including endochondral bone morphogenesis. These structurally related proteins have been identified as being involved in a variety of developmental events. For example, TGF-xcex2 and the polypeptides of the inhibin/activin group appear to play a role in the regulation of cell growth and differentiation. MIS causes regression of the Mullerian duct in development of the mammalian male embryo, and dpp, the gene product of the Drosophila decapentaplegic complex, is required for appropriate dorsal-ventral specification. Similarly, Vg-1 is involved in mesoderm induction in Xenopus, and Vgr-1 has been identified in a variety of developing murine tissues. Regarding bone formation, proteins in the TGF-xcex2 superfamily, for example OP-1 and a subset of other proteins identified as BMPs (bone morphogenic proteins) play the major role. OP-1 (BMP-7) and other osteogenic proteins have been produced using recombinant techniques (U.S. Pat. No. 5,011,691 and PCT Application No. US 90/05903) and shown to be able to induce formation of true endochondral bone in vivo. The osteogenic proteins generally are classified in the art as a subgroup of the TGF-xcex2 superfamily of growth factors (Hogan (1996), Genes and Development, 10:1580-1594).
Recently certain members of this same family of proteins have been recognized to be morphogenic, i.e., capable of inducing the developmental cascade of tissue morphogenesis in a mature mammal (See PCT Application No. US 92/01968). In particular, these morphogens are capable of inducing the proliferation of uncommitted progenitor cells, and inducing the differentiation of these stimulated progenitor cells in a tissue-specific manner under appropriate environmental conditions. In addition, the morphogens are capable of supporting the growth and maintenance of these differentiated cells. These morphogenic activities allow the proteins to initiate and maintain the developmental cascade of tissue morphogenesis in an appropriate, morphogenically permissive environment, stimulating stem cells to proliferate and differentiate in a tissue-specific manner, and inducing the progression of events that culminate in new tissue formation. These morphogenic activities also allow the proteins to induce the xe2x80x9credifferentationxe2x80x9d of cells previously stimulated to stray from their differentiation path. Under appropriate environmental conditions it is anticipated that these morphogens also may stimulate the xe2x80x9credifferentiationxe2x80x9d of committed cells.
Members of this morphogenic class of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF-3 (also known as Vgr2), GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2 or BMP-13), GDF-7 (also known as CDMP-3 or BMP-12), the Xenopus homolog Vg1 and NODAL, UNWIN, SCREW, ADMP, and NEURAL, to name but a few.
By way of illustration using exemplary family members, the tertiary and quaternary structure of both TGF-xcex22 and OP-1 have been determined. Although TGF-xcex22 and OP-1 exhibit only about 35% amino acid identity in their respective amino acid sequences the tertiary and quaternary structures of both molecules are strikingly similar. Both TGF-xcex22 and OP-1 are dimeric in nature and have a unique folding pattern involving six of the seven C-terminal cysteine residues. In each subunit four cysteines bond to generate an eight residue ring, and two additional cysteine residues form a disulfide bond that passes through the ring to form a knot-like structure. With a numbering scheme beginning with the most N-terminal cysteine of the 7 conserved cysteine residues assigned number 1, the 2nd and 6th cysteine residues bond to close one side of the eight residue ring while the 3rd and 7th cysteine residues close the other side. The 1st and 5th conserved cysteine residues bond through the center of the ring to form the core of the knot. The 4th cysteine forms an interchain disulfide bond with the corresponding residue in the other subunit.
The TGF-xcex22 and OP-1 monomer subunits comprise three major structural elements and an N-terminal region. The structural elements are made up of regions of contiguous polypeptide chain that possess over 50% secondary structure of the following types: (1) loop, (2) xcex1-helix and (3) xcex2-sheet. Furthermore, in these regions the N-terminal and C-terminal strands are not more than 7 xc3x85 apart. The residues between the 1st and 2nd conserved cysteines form a structural region characterized by an anti-parallel xcex2-sheet finger, referred to herein as the finger 1 region (F1). Similarly the residues between the 5th and 6th conserved cysteines also form an anti-parallel xcex2-sheet finger, referred to herein as the finger 2 region or sub-domain (f2). A xcex2-sheet finger is a single amino acid chain, comprising xcex2-strand that folds back on itself by means of a xcex2-turn or some larger loop so that the entering and exiting strands forth one or more anti-parallel xcex2-sheet structures. The third major structural region, involving the residues between the 3rd and 4th conserved cysteines is characterized by a three turn xcex1-helix referred to herein as the heel region (H). In the dimeric forms of both TGF-xcex22 and OP-1, the subunits are oriented such that the heel region of one subunit contacts the finger regions of the other subunit with the knot regions of the connected subunits forming the core of the molecule. The 4th cysteine forms a disulfide bridge with its counterpart on the second chain thereby equivalently linking the chains at the center of the palms. The dimer thus formed is an ellipsoidal (cigar shaped) molecule when viewed from the top looking down the two-fold axis of symmetry between the subunits.
Whether naturally-occurring, or recombinantly or synthetically prepared, true morphogens within the TGF-xcex2 superfamily, such as osteogenic proteins, can induce recruitment and stimulation of progenitor cells, thereby inducing their differentiation, e.g., into chondrocytes and osteoblasts, and further inducing differentiation of intermediate cartilage, vascularization, bone formation, remodeling, and, finally, marrow differentiation. Numerous practitioners have demonstrated the ability of osteogenic proteins, when admixed with either naturally-sourced matrix materials such as collagen or synthetically-prepared polymeric matrix materials, to induce true bone formation, including endochondral bone formation, under conditions where true replacement bone would not otherwise occur. For example, when combined with a matrix material, these osteogenic proteins induce formation of new bone in large segmental bone defects, calvarial defects, spinal fusions, and fractures, to name but a few.
Bacterial and other prokaryotic expression systems are relied on in the art as preferred means for generating both native and biosynthetic or recombinant proteins. Further, total chemical synthesis is an emerging possibility for producing small proteins. Prokaryotic systems such as E. coli are useful for producing commercial quantities of proteins, as well as for evaluating biological and chemical properties of naturally occurring or synthetically prepared mutants and analogs. Typically, an over-expressed eukaryotic protein aggregates into an insoluble intracellular precipitate (xe2x80x9cinclusion bodyxe2x80x9d) in the prokaryote host cell. The aggregated protein is then collected from the inclusion bodies, solubilized using one or more standard denaturing agents, and then allowed, or induced, to refold into a functional state. Chemically synthesized proteins would also require proper refolding.
Optimal refolding requires proper formation of any disulfide bonds to form and stabilize a biologically active protein structure. However, not all naturally-occurring proteins when solubilized readily refold. Despite careful manipulation of the chemistries for refolding, the yields of optimally folded, stable and/or biologically active protein remain low. Many of the aforementioned proteins, including BMPs, fall into the category of poor refolder proteins. For example, while some members of the BMP protein family can be folded relatively efficiently in vitro as, for example, when produced in E. coli or other prokaryotic hosts, many others, including BMP-5, BMP-6, and BMP-7, are not. See, e.g., EP 0433225 and U.S. Pat. No. 5,399,677, U.S. Pat. No. 5,756,308, and U.S. Pat. No. 5,804,416.
A need remains for improved means for designing and successfully producing recombinant, chemosynthetic and/or biosynthetic members of the TGF-xcex2 superfamily of proteins, including morphogenic proteins.
The present invention provides modified proteins, and DNAs encoding the same, of the TGF-xcex2 superfamily including morphogenic proteins. As used herein, the terms xe2x80x9cmodified TGF-xcex2 superfamilyxe2x80x9d, xe2x80x9cmutant TGF-xcex2 superfamilyxe2x80x9d, xe2x80x9cmutant proteinxe2x80x9d, xe2x80x9cmutant constructxe2x80x9d, and xe2x80x9cmutantxe2x80x9d refer to any TGF-xcex2 superfamily member synthetic construct wherein specific amino acids have been substituted by other amino acids or where any or all of a finger 2 sub-domain of one TGF-xcex2 superfamily member is replaced by any or all of a finger 2 sub-domain of another TGF-xcex2 superfamily member. Also contemplated herein, mutant proteins comprise recombinant and/or biosynthetic and/or chemosynthetic proteins. Additionally, mutant proteins of the invention have altered refolding attributes relative to naturally occurring proteins. Mutant proteins of the invention can have altered stability, specific activity, solubility, bioactivity and/or biospecificity attributes, and are useful for tissue-specific targeting.
In one embodiment, the invention provides synthetic mutant proteins that improve the refolding properties of the native xe2x80x9cpoor refolderxe2x80x9d proteins. As used herein, a xe2x80x9cpoor refolderxe2x80x9d protein means any protein that, when induced to refold under typically suitable refolding conditions, yields less than about 1% optimally refolded material (see below). Specifically, poor refolders are transformed to good refolders by specific amino acid sequence changes within the poor refolder""s C-terminal finger 2 sub-domain. The poor refolder protein""s folding capabilities also are enhanced by increasing the number of hydrophilic residues, particularly acidic (negatively charged) residues, in the protein""s finger 2 sub-domain.
Increasing the number of acidic residues in the base region of the finger 2 sub-domain improves the refolding properties of the substituted sequence as compared with the refolding properties of the unsubstituted parent sequence. The poor refolder protein""s folding capabilities also are enhanced by increasing the number of hydroxyl-group carrying polar residues in the protein""s finger 2 sub-domain, particularly in the base region of the finger 2 sub-domain. Further, it has been discovered that individual sub-domains of the C-terminal active region of a TGF-xcex2 superfamily member protein can be exchanged to acquire a desired biological or biochemical property.
In a preferred embodiment, pail or all of the finger 2 base region of a poor refolder is exchanged for the finger 2 base region of a good refolder to improve the folding properties of the parent protein without substantially affecting the biological specificity of the TGF-xcex2 superfamily member protein. It is anticipated that the receptor binding specificity of a given TGF-xcex2 superfamily member protein is altered by exchanging the finger 2 tip region of one TGF-xcex2 superfamily member protein with that of a different TGF-xcex2 superfamily member protein without substantially affecting the folding properties of the parent protein.
As a result of these discoveries, means are now available for predicting and designing de novo TGF-xcex2 superfamily member mutants having altered attributes, including altered folding capabilities, altered solubility, altered stability, altered isoelectric points, altered surface or carrier binding properties, and/or altered biological activities and specificities, as desired. The invention also provides means for easily and quickly evaluating biological and/or biochemical attributes of chimeric constructs, including mapping epitopes. In particular, candidates are constructed with particular specific amino acid sequences in the finger 2 sub-domain, thereby allowing expression of the candidate sequence in a prokaryotic host, such as E. coli, followed by refolding in vitro.
As used herein, a xe2x80x9cpoor refolderxe2x80x9d protein means any protein that, when induced to refold under typically suitable refolding conditions, yields less than about 1% optimally refolded material, as measured using a standard protocol. As contemplated herein, xe2x80x9ctypically suitable refolding conditionsxe2x80x9d are conditions under which proteins can be refolded to the extent required to confer functionality. One skilled in the an will recognize that at least Section I.B.1.(c) and Example 3 disclose non-limiting examples of such refolding conditions. Structural parameters relevant to the compositions and methods of the instant invention include one or more disulfide bridges properly distributed throughout the dimeric protein""s structure and which require a reduction-oxidation (xe2x80x9credoxxe2x80x9d) reaction step to yield a folded structure. Redox reactions typically occur at neutral pHs. Accordingly, as used herein, xe2x80x9csuitable refolding conditionsxe2x80x9d include a redox reaction step at a substantially neutral pH, i.e., in the range of about pH 5.0-10.0, typically in the range of about pH 6.0-9.0, and preferably under physiologically compatible conditions. The skilled artisan will appreciate and recognize optimal conditions for success.
In one preferred embodiment, the invention provides recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins having altered refolding properties under neutral or otherwise physiologically-compatible conditions. By way of example only, the recombinant, chemosynthetic or biosynthetic proteins of the invention have altered refolding properties at a pH in the range of about 5.0-10.0, preferably in the range of about 6.0-9.0, more preferably in the range of about 7.0-8.5, including in the range of about pH 7.5-8.5.
In another embodiment, the invention provides recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins having altered solubility attributes under neutral or otherwise physiologically compatible conditions. In one embodiment, the proteins of the invention have altered solubility at a pH in the range of about 5.0-10.0, preferably in the range of about 6.0-9.0, more preferably in the range of about 6.0-8.5, including in the range of about pH 7.0-7.5. In another embodiment, the stability of a protein is altered by the modifications and manipulations disclosed herein. xe2x80x9cAlteredxe2x80x9d is intended to mean different from the native protein""s attribute(s), and includes embodiments which can be more stable or less stable, and/or more soluble or less soluble, etc.
In still another embodiment, the invention provides recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins competent to refold under physiologically compatible conditions and having altered isoelectric points as compared with the native protein. In another embodiment, the invention provides recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins having altered refolding properties as compared with the native protein, and wherein the mutant proteins also have, for example, altered receptor binding specificity, altered stability, altered solubility and/or biological activity.
In another aspect, the invention provides recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins having improved refolding properties as compared with the parent protein, and, in certain preferred embodiments, wherein at least one residue, preferably two, more preferably three or more residues, in the neck or base region of a poor refolder protein""s finger 2 sub-domain is replaced with an acidic residue. In certain other embodiments more than five residues are replaced. In one embodiment the substituting acidic residue is Glu or Asp. In another preferred embodiment, the acidic residue replaces a basic residue or an amide group-carrying hydrophilic residue in the base region. In still another preferred embodiment the parent protein is any one of OP-1 BMP-5, BMP-6, OP-2, OP-3 or 60-A, including chimerics, heterodimers and amino acid variants thereof, and the substitution with an acidic residue is made at least at one of the following positions 4(Q), 6(N), 25(R), 26(N), 30(R), and potentially also at 22(K), 23(K) and 31(A). Counting from the first residue following the cysteine doublet in the C-terminal domain of OP-1. (See e.g., FIG. 1). In another embodiment the parent protein is any one of NODAL, TGF-xcex21, TGF-xcex22, TGF-xcex23, TGF-xcex24 and TGF-xcex25, and the substitution is made at one or more of the following positions, 4(K, D, T, A, V), 6 (K, E, D), 24 (K, S), 25 (D, N), 29 (E, K, R) and possibly 30 (E, S, A).
In another aspect, the invention provides recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins having improved refolding properties as compared with the parent protein, and, in certain preferred embodiments, wherein at least one residue, preferably two, more preferably three or more residues, in the neck or base region of a poor refolder protein""s finger 2 sub-domain is replaced with a hydroxyl group-carrying polar residue. In certain other embodiments more than five residues are replaced. In one embodiment the substituting polar residue is Ser or Thr. In another preferred embodiment, the substituting residue replaces a basic residue or an amide group-carrying hydrophilic residue in the base region. In still another preferred embodiment the parent protein is any one of OP-1 BMP-5, BMP-6, OP-2, OP-3 or 60-A, including chimerics, heterodimers and amino acid variants thereof, and the substitution with an acidic residue is made at least at one of the following positions: 4, 6, 25, 26, 30, potentially 22, 23, and 31 from the first residue following the cysteine doublet in the C-terminal domain. (See e.g., FIG. 1). In another embodiment the parent protein is any one of NODAL, TGF-01, TGF-xcex22, TGF-xcex23, TGF-xcex24 and TGF-xcex25, and the substitution is made at one or more of the positions, 4, 6, 24, 25, 29 and potentially 30.
In another embodiment the substitution of residues with an acidic residue and/or hydroxyl group-carrying polar residue occurs at least at one of the following positions in OP-1 (SEQ ID NO: 39): 400, 402, 405, 421, 422, 426, corresponding to residues 4, 6, 9, 25, 26 and 30, respectively, when counting from the first residue following the conserved cysteine doublet in the C-terminal active domain (see FIG. 1).
In another aspect, the invention provides recombinant, chemosynthetic, or biosynthetic TGF-xcex2 superfamily member proteins, including heterodimers and chimeras, wherein the C-terminal residue has been replaced with any one of the amino acid residues: arginne, serene, isoleucine, leucine or alanine and having improved refolding properties as compared to the unsubstituted parent TGF-xcex2 superfamily member protein sequence. In one preferred embodiment, the TGF-xcex2 superfamily member is OP-1. In another embodiment the TGF-xcex2 superfamily member is any one of BMP-5, BMP-6, 60-A, or OP-2. In still another embodiment, the TGF-xcex2 superfamily member is any of GDF-3, SCREW or NODAL. In one preferred embodiment, the substituting residue is arginine. In another preferred embodiment the C-terminal residue in the parent sequence that is replaced is histidine. In another preferred embodiment, the parent protein is any poor refolder protein included in the list of any one of OP-1, BMP-5, BMP-6, OP-2, OP-3, 60-A, NODAL, GDF-3 or SCREW, chimerics and/or amino acid variants thereof, and the substituting C-terminal residue is arginine. In another embodiment, the parent protein is any poor refolder protein included in the list of TGF-xcex21, TGF-xcex22, TGF-xcex23, TGF-xcex23 and TGF-xcex25, including chimeras and/or amino acid variants thereof, and the substituting C-terminal residue is arginine. In one embodiment these C-terminally substituted proteins further include one or more amino acid residue substitutions in the finger 2 sub-domain to increase the number of acidic (negatively charged) residues in this region to at least 3, preferably 4, more preferably 5. In a preferred embodiment, the acidic residue substitution is made in the neck or base region of the finger 2 sub-domain. In one embodiment the substituting acidic residue is Asp or Glu. In still another embodiment, the acidic residues replace hydrophilic residues, particularly amide groups-carrying and/or positively charged residues in this region. In still another embodiment, these replaced residues include: Asn, Gln, His, Arg and Lys. In another embodiment, the C-terminally substituted proteins further include one or more amino acid residue substitutions in the finger 2 sub-domain to reduce the number of amide group carrying residues and/or positively charged residues in this region. In one embodiment, the substituted finger 2 sub-domain has less than five positively charged residues, preferably 4 or less, more preferably 3 or less, positively charged residues in the base region of finger 2. In still another embodiment, the C-terminally substituted proteins further include one or more amino acid residue substitutions in the finger 2 sub-domain to increase the number of hydroxyl group carrying residues in this region. In one embodiment the substituting polar residue is Ser or Thr. In another embodiment the substituting residue replaces a basic or amide group-carrying residue.
Modified proteins of the invention can be used in conjunction with a biocompatible matrix such as collagen, hydroxyapatite, ceramics or carboxymethylcellulose, or other suitable matrix material. Such combinations are particularly useful in methods for regenerating bone, cartilage and/or other non-mineralized skeletal or connective tissues such as, but not limited to, ligament, tendon, muscle, articular cartilage, fibrocartilage, joint capsule, menisci, intervertabral discs, synovial membrane tissue, and fasica to name but a few. See e.g. U.S. Pat. Nos. 5,496,552, 5,674,292, 5,840,325 and U.S. Ser. No. 08/253,398, soon-to-issue as U.S. Pat. No. 5,906,827, the disclosures of which are incorporated by reference herein; also incorporated by reference herein are co-pending U.S. Ser. Nos. 08/459,129 and 08/458,811 each filed on Jun. 2, 1995. The instant application contemplates that the binding and/or adherence properties to such matrix materials can be altered using the specific mutations and techniques disclosed herein.
In another embodiment, the invention provides a method for folding those native TGF-xcex2 superfamily proteins which are poor refolders, such as BMP homodimers and heterodimers, as well as for folding the mutant proteins of the present invention under physiologically compatible and/or neutral pH conditions. In one preferred embodiment, the method comprises the steps of providing one or more solubilized substituted TGF-xcex2 superfamily member mutants of the invention, exposing the solubilized mutant to a redox reaction in a suitable refolding buffer, and allowing the protein subunits to refold into homodimers and/or heterodimers, as desired. In another embodiment, the redox reaction system can utilize oxidized and reduced forms of glulathione, DTT, xcex2-mercaptoethanol, xcex2-mercaptomethanol, cystine and cystamine. In another embodiment the redox reaction system relies on air oxidation, preferably in the presence of a metal catalyst, such as copper. In still another embodiment, ratios of reluctant to oxidant of about 1:10 to about 10:1, preferably in the range of about 1:2 to 2:1, can be used. In another preferred embodiment, the mutant protein is solubilized in the presence of a detergent, including a non-ionic detergent, e.g. digitonin N-octyl glucoside, or zwitterionic detergents, such as 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfate (CHAPS). In still another embodiment, the refolding reaction occurs in a pH range of about 5.0-10.0, preferably in the range of about 6.0-9.0, more preferably in the range of about 7.0-8.5. In still another embodiment, the refolding reaction occurs at a temperature within the range of about 0-32xc2x0 C., preferably in the range of about 4-25xc2x0 C. Where heterodimers are being created, optimal ratios for adding the two different subunits readily can be determined empirically and without undue experimentation.
Purification of heterodimers can be facilitated by adjusting the two monomeric sequences so that they differ in a property useful for purification, such as net charge.
Certain of the compositions of the present invention preferably are manufactured in accordance with the principles disclosed herein by assembly of nucleotides and/or joining DNA restriction fragments to produce synthetic DNAs. The DNAs are transfected into an appropriate protein expression vehicle, the encoded protein expressed, folded, and purified. Particular constructs can be tested for agonist activity in vitro. The tertiary structure of the candidate constructs can be iteratively refined and modulated by site-directed or nucleotide sequence directed mutagenesis aided by the principles disclosed herein, computer-based protein structure modeling, and recently developed rational molecular design techniques to improve or modulate specific properties of a molecule of interest. Known phage display or other expression systems can be exploited to produce simultaneously a large number of candidate constructs. The pool of candidate constructs subsequently can be screened for altered and/or improved binding specificity using, for example, a chromatography column comprising surface immobilized receptors, salt gradient elution to select for, and to concentrate high binding candidates, and in vitro assays to determine whether or not particular isolated candidates agonize the activity of the template superfamily member(s). Identification of a useful construct is followed by production of cell lines expressing commercially useful quantities of the construct far laboratory use and ultimately for producing therapeutically useful molecules. It is contemplated also that preferred constructs, once identified and characterized by the protein and DNA methodologies described herein, can be produced by standard chemical synthesis methodologies.
In another aspect, the invention provides methods for producing TGF-xcex2 superfamily member proteins in a host cell, including a bacterial host, or any other host cell where overexpressed protein aggregates in a form that requires solubilization and/or refolding in vitro. The method comprises the steps of providing a host cell transfected with nucleic acid molecules encoding one or more of the recombinant or biosynthetic proteins of the invention, cultivating the host cells under conditions suitable for expressing the recombinant protein, collecting the aggregated protein, and solubilizing and refolding the protein using the steps outlined above. In another embodiment, the method comprises the additional step of transfecting the host cell with a nucleic acid encoding the recombinant or biosynthetic protein of the invention.
In another aspect the invention provides recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins having little or no substantial increase in immunogenic effect in a mammal as compared with the parent sequence. The immunogenic effect of a molecule can be readily detected by injecting the molecule, or a portion thereof, into an animal, e,g., a test mouse, and assaying for antibodies produced by the animal in response to the injection and specific for the molecule. Useful assays include any standard protocol for detecting antibodies in serum, for example standard western blots, ELISA""s or radio immuno-assays.
In still another aspect the invention provides novel methods for creating recombinant, chemosynthetic or biosynthetic TGF-xcex2 superfamily member proteins having altered biological properties, including chimeric constructs. In one embodiment individual sub-domains of the C-terminal active region can be exchanged between TGF-xcex2 superfamily member molecules to create a chimeric construct with the desired property. For example, the finger 2 sub-domain of a poor refolder can be exchanged for the finger 2 sub-domain of a good refolder to improve the folding properties of the parent protein. Preferably, only non-conserved residues corresponding to the neck or base region finger 2 are exchanged and the loop or tip sequence of the parent sequence is maintained so as to maintain the biological activity of the parent sequence. Alternatively, the biological activity and the folding properties of a parent pour refolder protein can be altered by changing non-conserved residues in both the neck/base region and the tip/loop region of finger 2. Conversely, the biological activity of a good refolder can be altered, without substantially affecting the refolding properties of the protein by replacing the parent protein""s finger 1 sub-domain and/or heel sub-domain with that of a TGF-xcex2 superfamily member protein having the desired activity, thereby producing a mutant protein. Alternatively, just non conserved residues in the tip of finger 2 can be altered.